The granule exocytosis model of lymphocyte-mediated cytotoxicity postulates an antigen-triggered rapid secretion of the preformed potent lytic agent perforin (as well as other mediators) into the synapse-like space between the cytotoxic lymphocyte and its bound target. However, the question of why the effector cell does not itself die of perforin damage has often been raised without a clear answer. We have recently proposed that transiently expressed surface cathepsin B is provides such self protection to cytotoxic T cells and NK cells. Two major lines of evidence support this model: 1) When triggered to degranulate in the presence of membrane impermeant cathepsin B-specific inhibitors, cytotoxic lymphocytes undergo a rapid perforin-dependent, FasL-independent death; 2) A proteolytically active form of cathepsin B is expressed on the surface of cytotoxic T cells after degranulation. Since cathepsin B was previously shown by others to be expressed on the surface of tumor cells, we have tested whether it may be responsible for resisting CTL lethal damage in vitro. A series of in vitro tumor lines was screened to determine if pretreating them with membrane impermeant cathepsin B-specific inhibitors renders them more susceptible to redirected CTL-induced death. Most non-lymphoid tumors seem to be >10x less lysable than lymphoid tumors such as Jurkat after equivalent CTL degranulation. In some cases pre-treatment with CA074 causes a marked increase in lysability. We have seen this behavior with some but not all melanoma and colon tumor cells, and are currently screening a variety of available tumor lines. We previously showed that the cytoplasmic protein Rab27a was required for a late step in granule exocytosis in cytotoxic lymphocytes, but its functional molecular partners remain undefined. We are currently exploring immunoprecipitation-based approaches to identify a putative melanophilin homolog operating in CTL. Cathepsin W is a novel cathepsin originally described as an expressed sequence tag and subsequently found in mRNA expression studies to be expressed exclusively in cytotoxic lymphocytes (both T and NK cells). In order to study the protein and probe its function, we expressed human pre-procathepsin W in E. coli and made a series of monoclonal antibodies against it. In Western blots of CTL, some of these react with a single 25-30kd band, a size expected for active cathepsin W. When these mAb were used to localize cathepsin W, a granular pattern was seen by fluorescence microscopy. We are also examining the expression of granule proteins in naive and memory subpopulations of human blood T lymphocytes phenotypically defined by CCR7 and CD45RO expression. These studies show that memory subsets of both CD4+ and CD8+ express granule markers, which are minimally expressed in naive T cells.